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Abstract:

A method for determining a prognosis for survival for a patient with
leukaemia is described. Also described is a method for monitoring the
effectiveness of a course of treatment for a patient with leukaemia, and
the use of such a method in a kit. A kit determining the level of RINF is
also described.

Claims:

1. A method for determining the progression of the disease or a prognosis
for survival of a patient with acute myeloid leukaemia, the risk of a
relapse of acute myeloid leukaemia in a patient diagnosed and,
optionally, treated for acute myeloid leukaemia, characterized in that
said method comprising: a) determining the level of RINF expression in a
biological sample from said patient, b) comparing the level of RINF
expression in said sample to a reference level of RINF, whereby a level
of RINF expression in said sample of said patient lower than the
reference level correlates with better prognosis and increased survival
of said patient, slower progression of the disease or lower risk of a
relapse, and whereby a level of RINF expression in said sample higher
than the reference level correlates with decreased survival and bad
prognosis, faster progression of the disease or higher risk of relapse.

2. A method for the stratification of a patient being afflicted with
leukaemia to determine the therapy regimen for the treatment of leukaemia
comprising a) determining the relative level and/or the absolute amount
of RINF in a sample from said patient; b) comparing the level and/or
amount of RINF obtained in step a) to the level and/or amount of RNIF in
a control sample, with the level and/or amount of RINF in samples
obtained prior to the begin of the therapy of said patient, or obtained
in earlier stages of the therapy regimen of said patient.

3. (canceled)

4. The method according to claim 1, wherein the acute myeloid leukaemia
is acute myeloid leukaemia with normal karyotype.

6. The method according to claim 1 wherein the level of RINF is
determined as a relative level or an absolute amount.

7. The method according to claim 1 wherein the level and/or amount of
RINF is determined by quantification of the expression of mRNA encoding
for RINF or a quantification of a nucleic acid comprising a sequence of
SEQ ID No. 1 or SEQ ID No. 2 or a functional fragment or variant thereof,
or a functional equivalent isolated DNA sequence hybridisable thereto, or
a level of RINF expression is determined on protein level.

8. The method according to claim 7 wherein the level of RINF is
determined by quantitative RT-PCR whereby at least parts of SEQ ID No. 1
or SEQ ID No. 2 are amplified and/or wherein said RT-PCR uses primers
amplifying exon 3 and 4, or exon 1 and 2 of SEQ ID Nos. 1 to 3 and/or
wherein the level of RINF expression is determined based on the relative
expression of RINF normalized to the expression of the gene rpP2 also
known as RPLP2 (acidic ribosomal phosphor protein P2).

10. A method according to claim 1 wherein the level of RINF expression is
determined immunochemically, preferably based on an antibody-based
detection system.

11. The method according to claim 10 wherein said antibody binds
specifically to a protein of SEQ ID No. 3 or a fragment thereof,
preferably wherein said antibody binds specifically to the amino acids 45
to 48 of SEQ ID No. 3.

12. A method according to claim 1 wherein said method further comprises
the step c) classifying said patients as belonging to either a first or a
second group of patients, wherein said first group of patients having
levels of RINF expression lower than the reference level is classified as
having an increased likelihood of survival, slower progression of the
disease, better prognosis or lower risk or relapse compared to said
second group of patients having levels of RINF expression higher than the
reference level.

13. A method according to claim 1 wherein said method further comprises
the step c) classifying said patient as belonging to either a first or a
second group of patients, wherein said first group of patients having
levels of RINF expression lower than said second group of patients, and
where the first group of patients is classified as having an increased
likelihood of survival, slower progression of the disease, better
prognosis or lower risk or relapse compared to said second group of
patients.

14. The method according to claim 1 wherein when comparing the level of
RINF expression prior to the treatment with the level of RINF expression
after or during treatment indicates the effectiveness of said treatment
wherein a decrease in the level of RINF expression after treatment
indicates the treatment is effective against said acute myeloid
leukaemia.

15. The method according to claim 1 wherein the control sample or
reference sample is from an individual not afflicted with acute myeloid
leukaemia.

16. The method according to claim 2, wherein the stratification is
conducted based on the first diagnosis of acute myeloid leukaemia, during
initial treatment of acute myeloid leukaemia, after the first regimen of
treatment of acute myeloid leukaemia or during monitoring of acute
myeloid leukaemia relapse.

17. A kit for use in determining prognosis or survival, progression of,
or stratification of a patient with acute myeloid leukaemia,
characterized in that said kit comprises compounds capable of detecting
the level of RINF expression in a biological sample.

20. The kit according to claim 17 wherein said kit comprises antibodies
for determination of a level of RINF expression.

21. (canceled)

22. (canceled)

23. A use of means for determining RINF expression for stratification of
a patient being afflicted with acute myeloid leukaemia to determine the
therapy regimen for the treatment of acute myeloid leukaemia in said
individual.

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to a method for determining a
prognosis for survival or the progression of the disease for a patient
with leukaemia, the risk of resistance to treatment, or the risk of a
relapse of leukaemia, like an acute myeloid leukaemia, e.g. an acute
myeloid leukaemia with normal karyotype or a CBF (Core-Binding-Factor)
acute myeloid leukaemia, and a method for monitoring the effectiveness of
a course of treatment for a patient with leukaemia, as well as a kit for
use in said methods, preferably, by quantifying RINF mRNA expression.

BACKGROUND FOR THE INVENTION

[0002] Identification of genes specifically deregulated in tumour cells
could bring to light new tumour suppressors and oncogenes with potential
clinical applications in diagnostics, prognostics, and therapeutics. In
PCT application WO 2009/151337 a new target gene of retinoids, CXXC5,
encoding a nuclear factor has been described that was functionally
characterized for the first time at the mRNA and protein level and named
RINF (Retinoid-Inducible Nuclear Factor). In PCT application WO
2010/085153 it is described for the first time that measurement of RINF
mRNA in breast cancer tumours, could be informative for overall survival
of breast cancer patients.

[0003] Present data indicate that RINF plays an essential role during in
vitro human hematopoiesis. Indeed, expression studies and gene silencing
experiments both demonstrate RINF implication during terminal
differentiation of myeloid leukaemia cells (NB4 and HL60 cell lines), but
also during myelopoiesis of normal hematopoietic progenitors (bone marrow
CD34+ cells). In keeping with its essential role during terminal myeloid
differentiation and its localization to chromosome 5q31.3, a region often
deleted in myeloid leukaemia (more especially in acute myeloid leukaemia
and myelodysplasia), RINF has been suggested as a promising tumour
suppressor candidate at that locus in some myeloid malignancies.

[0004] Because RINF expression is not restricted to the hematopoietic
tissue and may also be involved in development and/or homeostasis of
other tissues, RINF expression in some solid tumours derived from
different tissues have been investigated. It is shown in PCT application
WO 2009/151337 that this gene could have a deregulated expression in some
malignant tissues compared to their normal tissues of origin. Indeed,
RINF mRNA expression was examined in solid tumour samples from patients
suffering from breast cancer, and RINF expression was significantly
higher in breast tumours compared to normal breast tissues controls.

[0005] Further, in PCT application WO 2010/085153 it is shown that
measurement of RINF mRNA in breast cancer tumours, could be informative
for overall survival of these breast cancer patients. In this
application, it is also described a "kit" for detection of RINF mRNA by
quantitative RT-PCR.

[0007] The major forms of leukaemia can be divided into four main
categories. Myelogenous and lymphocytic/lymphoblastic leukaemias, each
have acute and chronic forms. The terms myelogenous or
lymphocytic/lymphoblastic denote the cell type involved. In addition,
within each category, distinct leukaemias are defined according to a
combination of morphology, immunophenotype, and cytogenetic features in
addition to clinical symptoms.

[0008] Acute leukaemia is a clonal disease of hematopoetic stem cells. For
example, in the United States more than half of all new leukaemias are
acute leukaemias. Said acute leukaemias can be differentiated into acute
myelogenous leukaemia (AML), also called acute non-lymphocytic leukaemia
and acute lymphocytic or acute lymphoblastic leukaemia (ALL) based on
whether hematopetic stem cells have been differentiated along the
lymphoid or myeloid lineage. In addition, chronic types of leukaemia are
differentiated into chronic myelogenous leukaemia (CML) or chronic
lymphocytic/lymphoblastic leukaemia (CLL).

[0009] Among leukaemia, acute myeloid leukaemia (AML) represents a
distinct group of disease with heterogeneous genetic basis,
pathophysiology, and prognosis. In the past, the karyotype of the AML
cells has emerged as the most important prognostic factor, and patients
can be classified into favourable, intermediate, and unfavourable
prognostic groups according to the number and the specific types of
chromosomal aberrations. However, almost half of all adult AML patients
present with a normal karyotype at diagnosis. These patients are usually
assigned to the intermediate prognostic group. More recently, mutations
in specific genes that allow the identification of prognostic subgroups
in cytogenetically normal (CN) AML have been described. Internal tandem
duplications (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene are
strong predictors of inferior outcome. Mutations in the nucleophosmin 1
(NPM1) gene lead to cytoplasmic localization of the NPM protein and are a
favourable prognostic factor, especially in patients lacking FLT3 ITD.
Other prognostically relevant alterations include mutations in the MLL
and CEBPA genes as well as overexpression of WT1, MN1, BAALC, ERG, and
EVI1(MECOM). However, in approximately 24% of CN-AML cases, none of the
aforementioned mutations can be detected. These findings illustrate that
CN-AML itself constitutes a heterogeneous disease and that there probably
are many genetic alterations in CN-AML to be discovered that affect
response to treatment and survival.

[0010] In the case of leukaemia, like ALL, AML, CLL and CML, and
especially in the case of AML and CN-AML, but also with CBF-AML, these
prognostic indicators are very important to help the physicians to
distinguish the group of patients that would more likely benefit from a
chemotherapy (usually, disease with a favourable prognosis) from patients
that (more probably) would not, and for instance, that should be
considered for autologous or allogenic stem cell transplantation (these
patients present usually with unfavourable prognosis and age below 60).
In other terms, prognostic factors are of very high potential for the
treatment decision making in these malignant haemopathies and additional
prognostic markers are needed.

SUMMARY OF THE PRESENT INVENTION

[0011] The inventors have now surprisingly found that the level of RINF
expression correlates with the survival of patients with leukaemia, the
progression of the disease, the risk of relapse of leukaemia more
precisely an acute myeloid leukaemia, more precisely an acute myeloid
leukaemia with normal karyotype.

[0012] The present invention thus relates to methods for determining the
prognosis for survival, the progression of the disease, the risk of
relapse of leukaemia, and for testing the effect of treatment regimens.
The invention also relate to a kit for determining the prognosis for
survival, and for testing the effect of treatment regimens.

[0014] The microarray dataset (Affymetrix GeneChip Human Genome HG-U133B)
performed by Metzeler K H, et al., Blood. 2008 Nov. 15; 112(10):4193-201
(n=163 patient samples) was downloaded from the Gene Expression Omnibus
website (http://www.ncbi.nlm.nih.gov/geo/) with accession number
GSE12417. The whole raw data were normalized using RMA (Robust Multiarray
Averaging method) with the Expression Console software from Affymetrix.
The CXXC5 mRNA expression was then calculated as the mean intensity of
the 3 probesets targeting CXXC5 (222996_s_at, 224516_s_at and
233955_x_at). The patients have been classified in two equivalent groups
(A), according to an expression above (High, n=81) or below (Low, n=81)
the median, or in 3 equivalent groups (B and C), according to a High
(n=55), an Intermediary (n=54), or a Low (n=54) RINF expression level.
For panel C, the Low (n=54) and Intermediary (n=54) groups have been
fused. Note that because of an odd number of patients (n=163), the
patient at the median has been censored for the 2 groups stratification
and were not exactly equivalents for the 3 groups stratification (n=55
versus 54). The patient groups with high or low CXXC5 mRNA (RINF) were
then correlated with the clinical outcomes concerning survival that were
also accessed through the Gene Expression Omnibus website. The
Kaplan-Meier curves (for survival analysis) and the log-rank test were
performed by using the statistical SPSS 15.0. P values (log-rank test) of
the comparison of the various groups of patients are indicated in the
panels.

[0016] The microarray datasets (Standford microarrays) performed by
Gaidzik V I, et al., J Clin Oncol. 2011 Apr. 1; 29(10):1364-72 (n=269,
but n=266 patient samples with CXXC5 expression data available) was
downloaded from the Gene Expression Omnibus website
(http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE23312.
Fluorescence ratios were normalized by mean-centering genes for each
array and then mean-centering each gene across arrays. The CXXC5 mRNA
expression was then extracted and patients have been classified in three
equivalent groups (A and B), according to a High (n=88), an Intermediary
(n=89), or a Low (n=89) RINF expression level. For panel B, the High
(n=88) and Intermediary (n=89) groups have been fused (n=177). Note that
the patient number were not exactly equivalents for the 3 groups
stratification (n=89, 89 versus 88) since the total number of patients
was not a multiple of 3 (n=266). The patient groups with high,
Intermediary, and low CXXC5 mRNA were then correlated with the clinical
outcomes concerning survival that were also accessed through the Gene
Expression Omnibus website. The Kaplan-Meier curves (for survival
analysis) and the log-rank test were performed by using the statistical
SPSS 15.0. P values (log-rank test) of the comparison of the various
groups of patients are indicated in the panels.

[0017] FIG. 3: Low RINF mRNA expression is related to a better prognosis
in AML with normal karyotype (n=102). The microarray datasets (Standford
microarrays) performed by Gaidzik V I et al (see above) was used and
analysed as described above. The CXXC5 mRNA expression was then extracted
and patients have been classified in three equivalent groups (A and B),
according to a High (n=35), an Intermediary (n=34), or a Low (n=34) RINF
expression level. For panel B, the High (n=35) and Intermediary (n=34)
groups have been fused (n=69). Note that the patient number were not
exactly equivalents for the 3 groups stratification (n=34, 34 versus 35)
since the total number of patients was not a multiple of 3 (n=102). The
patient groups with High, Intermediary, and Low CXXC5 mRNA were then
correlated with the clinical outcomes concerning survival that were also
accessed through the Gene Expression Omnibus website. The Kaplan-Meier
curves (for survival analysis) and the log-rank test were performed by
using the statistical SPSS 15.0. P values (log-rank test) of the
comparison of the various groups of patients are indicated in the panels.

[0018] FIG. 4: Low RINF mRNA expression is related to a better prognosis
in AML with inversion (16) (n=31). The microarray datasets (Standford
microarrays) performed by Gaidzik V I et al. was obtained and analysed as
described above. The CXXC5 mRNA expression was then extracted and
patients have been classified in three equivalent groups (A and B),
according to a High (n=11), an Intermediary (n=10), or a Low (n=10) RINF
expression level. For panel B, the High (n=11) and Intermediary (n=10)
groups have been fused (n=21). Note that the patient number were not
exactly equivalents for the 3 groups stratification (n=10, 10 versus 11)
since the total number of patients was not a multiple of 3 (n=31). The
patient groups with high, Intermediary, and low CXXC5 mRNA were then
correlated with the clinical outcomes concerning survival that were also
accessed through the Gene Expression Omnibus website. The Kaplan-Meier
curves (for survival analysis) and the log-rank test were performed by
using the statistical SPSS 15.0. P values (log-rank test) of the
comparison of the various groups of patients are indicated in the panels

[0019] FIG. 5: Low RINF mRNA expression is related to a better prognosis
in AML with translocation t(8;21) (n=19). The microarray datasets
(Standford microarrays) performed by Gaidzik V I et al. (n=269, but n=19
patient samples with CXXC5 expression data available and translocation
t(8;21)) was analysed as described above. The CXXC5 mRNA expression was
then extracted and patients have been classified in three equivalent
groups (A and B), according to a High (n=7), an Intermediary (n=6), or a
Low (n=6) RINF expression level. For panel B, the Low (n=6) and
Intermediary (n=6) groups have been fused (n=12). Note that the patient
number were not exactly equivalents for the 3 groups stratification (n=6,
6 versus 7) since the total number of patients was not a multiple of 3
(n=19). The patient groups with high, Intermediary, and low CXXC5 mRNA
were then correlated with the clinical outcomes concerning survival that
were also accessed through the Gene Expression Omnibus website. The
Kaplan-Meier curves (for survival analysis) and the log-rank test were
performed by using the statistical SPSS 15.0. P values (log-rank test) of
the comparison of the various groups of patients are indicated in the
panels

[0020] FIG. 6: FIG. 6 relates to the survival rate for marker molecules
described in the art as suitable marker molecules in leukaemia. The
cohort was classified in two or three groups, respectively, based on the
expression levels of the respective marker. (A) ERG, MECOM BAALC, (B)
MN1, WT1.

[0021] FIG. 7: In FIG. 3 RINF mRNA expression (at a diagnosis time) is
higher in the group of acute lymphocytic leukaemia patients (n=197) that
relapse after treatment than in persons showing no relapse.

[0023] In a first aspect, the present invention relates to a method for
determining the progression of the disease or a prognosis for survival of
a patient with leukaemia, the risk of a relapse of leukaemia in a patient
diagnosed and, optionally, treated for leukaemia, characterized in that
said method comprising:

[0024] a) determining the level of RINF
expression in a biological sample from said patient,

[0025] b) comparing
the level of RINF expression in said sample to a reference level of RINF,
whereby a level of RINF expression in said sample of said patient lower
than the reference level correlates with better prognosis and increased
survival of said patient, slower progression of the disease or lower risk
of a relapse, and whereby a level of RINF expression in said sample
higher than the reference level correlates with decreased survival and
bad prognosis, faster progression of the disease or higher risk of
relapse.

[0026] As used herein, the term "subject" or "individual" or "patient"
which is used herein interchangeably refers to an individual or a subject
or a patient in need of a therapy or suspected to be afflicted with a
condition or disease, namely leukaemia. Preferably, the subject or
individual is a vertebrate even more preferred a mammal, particularly
preferred a human.

[0027] The method according to the present invention is preferably
conducted by collecting a sample from the individual and determining in
vitro the level of RINF using appropriate means.

[0028] The term "control sample" or "reference sample" as used herein
refers e.g. to a sample of an individual of the same species which
individual is not suffering from leukaemia. Alternatively, the "control
sample" or "reference sample" is a sample representing the median of a
panel of patients afflicted with leukaemia. In another embodiment, the
"control sample" is a cell line sample (for instance such as NB4, HL60,
K562, MCF7).

[0029] A sample according to the invention is a biological material, which
has been obtained from an individual. The term "sample" and "biological
sample" are used herein interchangeably. In particular, said terms
include material obtained from blood, plasma, tissue, bone marrow, serum
or tumour, tumour biopsy or neoplastic cell containing sample.

[0030] The expression "level" or "amount" is meant to describe the
relative level of said molecules according to the present invention
relative to the same molecule in a control sample or reference sample, or
the absolute amount of said molecules according to the present invention.
Relative means that no distinct amounts such as mole or milligram per
litre etc. are stated, but that for example is stated the sample contains
more, less or the same amount of a certain molecule as compared to a
control sample. The term "more, less or the same amount" in this
situation includes also arbitrary units.

[0031] With "elevated" or "higher" and on the other side "decreased" or
"lower" according to the invention is meant, that the molecule is present
in a sample in higher (or lower, respectively) quantities, or amounts or
concentration, as compared to another sample, or as compared to a control
sample or as compared to a reference sample or as compared to a reference
value, regardless if these measurements are relative or absolute
measurements. Preferably, the molecule which is increased or decreased is
present in a concentration, or quantity or amount which is at least 10%,
like at least 20%, e.g. at least 30%, or 50% above or below the value to
which it is compared. In other words, preferably, the molecule is
increased or decreased at least 2 fold, e.g. 3 fold, 4 fold or even 5
fold or more above or below the level and/or amount in the control or
reference sample. For example, the molecule in the sample is higher than
4.5 fold time the level of NB4 cells measured in the reference sample.

[0032] With "stratification of a subject" according to the invention is
meant to determine the therapy regimen for the treatment of leukaemia. In
particular, said stratification includes determining whether said subject
will benefit from a specific treatment, irradiation chemotherapy, or stem
cell transplantation.

[0033] Prognosis is a medical term to describe the likely outcome of an
illness. Here, a good "prognosis for survival" correlates with high rate
survival of the patients and "bad prognosis for survival" correlates with
a low rate of survival of the patient group.

[0034] Thus, in one aspect, the present invention relates to a method for
determining a prognosis for survival for a patient with leukaemia,
wherein said method comprising:

[0035] (a) determining a measure for the level of RINF expression in a
biological sample from said patient,

[0036] (b) comparing the level of RINF expression in said sample to a
reference level of RINF,

wherein a level of RINF expression lower than the reference level in said
sample correlates with increased survival probability or rate of said
patient, and wherein a level of RINF expression higher than the reference
level in said sample correlates with decreased survival probability or
rate.

[0037] In this connection, the terms "a measure for the level" and "level"
are used interchangeably herein.

[0038] In a preferred embodiment, said biological sample is whole blood,
serum, plasma, liquor, bone marrow, a tumour, or a tumour biopsy or
neoplastic cell containing sample. In fact, the biological sample can be
a fraction of blood or bone marrow. for instance, it is a preferably PBMC
(peripheral blood mononuclear cells) or BMMC (bone marrow mononuclear
cells), isolated after ficoll-density gradient separation, and/or CD34+
cell sorting enrichment.

[0039] A preferred embodiment relates to acute myeloid leukaemia (AML).
Moreover, it is preferred that the leukaemia is a leukaemia with normal
karyotype or CBF, more preferable an acute myeloid leukaemia with normal
karyotype.

[0040] In a preferred embodiment said measure for the level of RINF
expression is determined by quantification of the expression of mRNA
which encodes for a RINF protein, or a quantification of a nucleic acid
comprising the sequence of SEQ ID NO1 or SEQ ID NO 2, or a functional
fragment or variant thereof, or a functionally equivalent isolated DNA
sequence hybridizable thereto.

[0041] In this connection, the term "hybridizable thereto" refers to
conditions wherein sequences hybridize under stringent hybridization
conditions (see, T. Maniatis et al., Cold Spring Harbor Laboratory
(1982), molecular Cloning/ a laboratory manual, pages 387 to 389) to the
sequences of Seq ID. No. 1 or 2. An example of stringent hybridization
condition is hybridization at 4×SSC at 65° C., followed by a
washing in 0.1×SSC at 65° C. for an hour. Alternatively, an
exemplary stringent hybridization condition is in 50% formamide,
4×SSC at 42° C.

[0042] Further, the term "functional fragment" refers to fragments of Seq.
ID. Nos. 1 or 2 having the same functionality as the polypeptide encoded
by Seq. ID. Nos. 1 or 2, respectively. Moreover, the term "variant"
refers to molecules having the same functionality as the nucleic acid
molecules of Seq. ID Nos. 1 and 2 or the polypeptides encoded by said
sequences, like the polypeptide of Seq. ID. No.3.

[0043] In a further preferred embodiment is the level of RINF expression
relative to said reference level used to determine a proper course of
treatment for said patient.

[0044] In a preferred embodiment is the level of RINF expression
determined by quantitative RT-PCR. More preferable, said quantitative
RT-PCR uses oligonucleotides targeting SEQ ID NO 1 or SEQ ID NO 2 for
determining the RINF expression, and more preferable said RT-PCR uses
primers amplifying exon 3 and 4, or exon 1 and 2 of SEQ ID NO 3.

[0045] Preferable, the level of RINF expression is determined based on the
relative expression of RINF normalized to the expression of the rpP2
gene, also known as RPLP2 ribosomal protein, large, P2, (HGNC: 10377).
Preferable, the primers for the detection of SEQ ID NO 1 or 2 are

[0046] 6-FAM is a reactive water-soluble fluorescent dye. It is an isomer
of carboxyfluorescein, widely used to label oligonucleotides for
real-time PCR applications. 6-FAM is excited maximally at 492 nm and
emits maximally at 517 nm.

[0047] Cy5 (Cyanine-5) is a reactive water-soluble fluorescent dye of the
cyanine dye family. Cy5 is excited maximally at 649 nm and emits
maximally at 670 nm, in the far red part of the spectrum.

[0048] BBQ means BlackBerry Quencher is a "dark quencher" for every
fluorescent dye. Non-fluorescent quenchers are fundamental for multiplex
applications with more than one dye. A dark quencher is a substance that
absorbs excitation energy from a fluorophore and dissipates the energy as
heat (while a typical (fluorescent) quencher re-emits much of this energy
as light).

[0049] Of course, any other marker or label may be used for effecting the
analysis accordingly. The skilled person is well aware of suitable label
or marker molecules.

[0050] Preferable, the PCR amplification is conducted by an initial
denaturation at 95° C. for 5 min, and then the samples run through
44 cycles of the following conditions: denaturation for 10 seconds at
95° C. and elongation at 55° C. for 20 seconds.

[0051] In a further embodiment the level of RINF expression is determined
immunochemically based on an antibody-based detection system. Said
antibody can be a polyclonal antibody, and is more preferable a
monoclonal antibody. Preferable, said antibody is interacting with an
epitope of a protein or protein sequence of SEQ ID NO 3, and more
preferable said antibody is interacting with the amino acids 45-48 of SEQ
ID NO 3.

[0052] In a further embodiment, the RINF expression is measured by a DNA,
RNA or protein array.

[0055] Among other Northern Blot or Southern Blot hybridization, nucleic
acid dot or slot Blot Hybridization, in situ hybridization, nucleic acid
chip assays (for example using RNA, DNA or other nucleic acids to
specifically capture the nucleic acid of interest), polymerase chain
reaction (PCR), reverse transcriptase-PCR (RT-PCR), real time PCR, and in
particular, real time quantitative polymerase chain reaction (RQ-PCR)
after reverse transcription may be used as molecular biology methods to
detect the molecules of the invention.

[0056] Further methods, such as nuclear magnetic resonance (NMR),
fluorometry, colorimetry, radiometry, luminometry or other spectrometric
methods, liquid chromatography, capillary chromatography, thin-layer
chromatography, plasmon resonance, one-or-two gel dimensional
electroporesis etc. can be used to detect the molecules of the invention.

[0057] In another preferred embodiment the level and/or amount of RINF is
determined on mRNA level. Particular preferred the determination is
conducted by RQ-PCR, for example applying known real time PCR-methods.
Another preferred embodiment relates to the determination of the level
and/or amount of RINF on protein level. For example, said level may be
determined by immunological methods as outlined above using specific
anti-RINF antibodies.

[0058] In a further embodiment, said method further comprises a step (c);

(c) classifying said patient as belonging to either a first or a second
group of patients, wherein said first group of patients having levels of
RINF expression lower than the reference level is classified as having an
increased likelihood of survival, slower progression of the disease,
better prognosis or lower risk or relapse compared to said second group
of patients having levels of RINF expression higher than the reference
level.

[0059] In a further embodiment, said method further comprises a step (c);

(c) classifying said patient as belonging to either a first or a second
group of patients, wherein said first group of patients having levels of
RINF expression lower than said second group of patients, and where the
first group of patients is classified as having an increased likelihood
of survival, slower progression of the disease, better prognosis or lower
risk or relapse compared to said second group of patients.

[0060] A second aspect of the present invention relates to a method for
monitoring the effectiveness of a course of treatment for a patient with
leukaemia, wherein said method comprising:

(a) determining a level of RINF expression in a biological sample from
said patient prior to treatment, and (b) determining the level of RINF
expression in a neoplastic cell-containing sample from said patient after
treatment, whereby comparison of the level of RINF expression prior to
treatment with the level of RINF expression after treatment indicates the
effectiveness of said treatment, wherein a decrease in the level of RINF
expression after treatment indicates that treatment is effective against
said leukaemia.

[0061] In addition, the present invention relates to a method for the
stratification of the patient being afflicted with leukaemia to determine
the therapy regimen for the treatment of leukaemia comprising

[0062] a)
the relative level and/or the absolute amount of RINF in a sample from
said patient;

[0063] b) comparing the level and/or amount of RINF to the
level and/or amount of RNIF in a control sample, with the level and/or
amount of RINF in samples obtained prior to the begin of the therapy of
said patient, or obtained in earlier stages of the therapy regimen of
said patient.

[0064] A third aspect of the present invention relates to a use in
accordance with any of the method claims, wherein at least one detection
member for RINF is used in a kit for determining a prognosis for survival
for a patient with leukaemia.

[0065] A forth aspect of the present invention relates to a kit for
determining a prognosis for survival for a patient with leukaemia,
characterized in that said kit comprises compounds capable of detecting
the level of RINF expression in a biological sample.

[0066] Preferable embodiments for the second, third and forth aspects
(independently for each aspect) are indicated below:

[0067] said leukaemia is AML, ALL, CML, or CLL, preferably acute myeloid
leukaemia, and more preferable acute myeloid leukaemia with normal
karyotype or CBF (Core-binding-factor) (CBF-AML are characterized by
translocation t(8:21) or inversion inv(16), respectively). CBF-AML
respond usually well to chemotherapy. However, some of these patients do
not respond well to therapy and it has been recognized that this groups
is characterized by high RINF expression.

[0068] said biological sample is obtained from bone marrow, blood or
tissue, like plasma, serum, liquor, a tumour, or a tumour biopsy or
neoplastic cell containing sample.

[0069] said measure for the level of RINF expression is determined by
quantification the expression of mRNA which encodes for a RINF protein,
or a quantification of a nucleic acid comprising the sequence of SEQ ID
NO1 or SEQ ID NO 2, or a functional fragment or variant thereof, or a
functionally equivalent isolated DNA sequence hybridizable thereto.

[0070] the level of RINF expression relative to said reference level is
used to determine a proper course of treatment for said patient.

[0071] the level of RINF expression is determined by quantitative RT-PCR.
More preferable, said quantitative RT-PCR uses oligonucleotides also
termed Hydrolysis Probes targeting SEQ ID NO 1 or SEQ ID NO 2 for
determining the RINF expression, and more preferable said RT-PCR uses
primers amplifying exon 3 and 4, or exon 1 and 2 of SEQ ID NO 3.

[0072] In this connection, the term "Hydrolysis probes" are used to define
sequence specific DNA oligonucleotides that are chemically modified
(labeled with both a fluorescent dye and a quencher dye) in order to
easily monitor the concentration of a specific DNA during a quantitative
real-time Polymerase Chain Reaction (qRT PCR).

The fluorescence emitted (when excited by an external light source at
each PCR cycle) is proportional to the amount of the specific DNA product
formed. The Hydrolysis Probe chemistry relies on the 5'-3' exonuclease
activity of Taq polymerase, which degrades a hybridized non-extendible
DNA probe during the extension step of the Polymerase Chain Reaction
(PCR). This probe is designed to specifically hybridize to a region
within the amplicon and is dual-labeled with a fluorecent dye and a
quencher. The close proximity of the quencher suppresses the fluorescence
of the reporter dye. Once the exonuclease activity of Taq polymerase
degrades the probe, the fluorescence of the reporter increases at a rate
that is proportional to the amount of template present.

[0073] the level of RINF expression is determined based on the relative
expression of RINF normalized to the expression of the rpP2 gene.

[0077] the PCR amplification is conducted by an initial denaturation at
95° C. for 5 min, and than the samples is run through 44 cycles of
the following conditions: denaturation for 10 seconds at 95° C.
and elongation at 55° C. for 20 seconds.

[0078] the level of RINF expression is determined immunochemically based
on an antibody. Said antibody can be a polyclonal antibody, and is more
preferable a monoclonal antibody. Preferable, said antibody is
interacting with an epitope of a protein or protein sequence of SEQ ID NO
3, and more preferable said antibody is interacting with the amino acids
45-48 of SEQ ID NO 3.

[0079] the RINF expression is measured by a DNA, RNA or protein array.

EXPERIMENTAL SECTION

Materials and Methods

Quantitative RT-PCR of RINF

[0080] First-strand cDNA synthesis (RT) was carried out starting with
total RNA (1 μg) in a 20 μl volume using oligo-dT primers with
Transcriptor Reverse Transcriptase (Roche, Cat. No 05 531 287 001) in
accordance with the manufacturer's instructions. Quantitative PCRs were
performed using specific Hydrolysis Probes targeting the CXXC5 gene on a
Light Cycler 480 machine (Roche) in accordance with the manufacturer's
instructions of the kit Lightcycler® 480 ProbesMaster (Cat. N°
04 707 494 001). Relative mRNA expressions were normalized to rpP2 gene
expression in a two-colour duplex reaction. Primers for detection of
CXXC5 (5'-tccgctgctctggagaag-3' (Seq. ID No. 4),
5'-cacacgagcagtgacattgc-3' (Seq. ID No. 5) and
6FAM-aacccaaagctgccctctcc-BBQ (Seq. ID No. 8)) and rpP2
(5'-atgcgctacgtcgcc-3' (Seq. ID No. 6), 5'-ttaatcaaaaaggccaaatcccat-3'
(Seq. ID No. 7) and Cy5-agctgaatggaaaaaacattgaagacgtc-BBQ (Seq. ID No.
9)), were designed to be used in the same conditions of real-time PCR
amplification. After initial denaturation at 95° C. for 5 min,
samples were run through 44 cycles of the following conditions:
denaturation for 10 seconds at 95° C. and elongation at 55°
C. for 20 seconds.

Analysis of CXXC5 mRNA Expression in a Microarray Dataset and Correlation
Analysis with the Overall Survival of Acute Myeloid Leukaemia Patients
with Normal Cytogenetics.

[0081] One microarray dataset for which (1) CXXC5 mRNA expression data,
and (2) survival data were available online, was downloaded from the Gene
Expression Omnibus website (http://www.ncbi.nlm.nih.gov/geo/) with
accession number GSE12417. This microarray dataset was originally
performed by Metzeler et al., see above in order to develop a gene
expression signature to predict treatment outcome for acute myeloid
leukaemia patients with normal karytotype (n=163 patient samples). For
each microarray platform (Affymetrix GeneChip® Human Genome U133A,
Affymetrix GeneChip® Human Genome U133B, and Affymetrix GeneChip®
Human Genome U133 Plus 2.0), the whole raw gene expression data were
normalized using RMA with the Expression Console software from
Affymetrix. The CXXC5 mRNA expression was calculated as the mean
intensity of the 3 probe sets targeting CXXC5 (222996_s_at, 224516_s_at
and 233955_x_at). Gene expression data were also extracted for some
already approved prognosis factors WT1 (206067_s_at and/or 206067_s_at),
MN1(205330_at), BAALC (222780_s_at), ERG (241926_s_at), MECOM (EVI,
226420_at). Patients were then clustered into two (above or below median)
or three (high, intermediary or low gene expression) groups according to
their CXXC5 mean intensity. The patient groups with high or low CXXC5
mRNA (RINF) were then correlated with the clinical outcomes concerning
survival that were also accessed through the Gene Expression Omnibus
website ("E-GEOD-12417.sdrf.xls" file). The Kaplan-Meier curves (for
survival analysis) and the log-rank test were performed by using the
statistical SPSS 15.0.

Detection of RINF by Antibody

[0082] The expression of RINF can also be determined by an antibody
approach. Western blotting was carried out as previously described by Wu
Y L, Dudognon C, Nguyen E, et al. Immunodetection of human telomerase
reverse-transcriptase (hTERT) m-appraised: nucleolin and telomerase cross
paths. J Cell Sci. 2006; 119:2797-2806.

[0083] Customized rabbit polyclonal peptide-specific antibody against RINF
was produced by Biogenes GmBH. The immunogenic peptide corresponds to
amino acids 45-58 of the RINF protein. Antibody specificity was confirmed
by competitive inhibition of the western-blot signal by addition of the
immunogene peptide to the primary antibody solution. Briefly, blots were
incubated with primary antibody against RINF (polyclonal antibody), and
then with an appropriate peroxydase conjugated secondary antibody.
Detection of protein was performed using a chemiluminescent detection
system (Amersham Pharmacia Biotech).

Kit

[0084] For the expression studies of the above mentioned gene/protein,
RINF, any method for quantitatively detecting the amplified product can
be used, including, for example using any fluorescent dyes or labeled
probes and standard methods.

[0085] Quantification of a sample containing an unknown number of target
sequences typically is carried out with reference to a "standard curve"
generated from a series of amplifications of samples containing the
target sequence in a range of known amount. The standard curve is used to
calculate an input copy number from the signal generated during an
amplification. Thus, the unknown target sequence copy number in the
sample of interest is estimated using the standard curve calculating the
copy number that previously was determined to yield a signal equal to
that observed. The concentration of the target sequence in the sample
then is calculated from the input copy number and the sample size, which
is determined prior to the reaction.

[0086] Quantitative estimates can be sensitive to variability in either
the input sample size or in the reaction efficiency. The effect of
inter-reaction variability of the input sample size on the calculated
RINF concentration can be eliminated by using a control gene. A control
gene is selected which provides an independent measure of the amount of
RNA in the sample. The calculated concentration of the RINF mRNA is
adjusted based on the independent measure of the sample.

[0087] Variability in the amplification efficiency between the reactions
used to generate the standard curve and the reaction used to assay the
sample of interest can affect the applicability of the standard curve.
Carrying out the reactions used to generate the standard curve
simultaneously with the reaction used to assay the sample of interest,
using the same "master mix" of amplification reagents, and, preferably,
in adjacent wells in the same thermal cycler, will minimize the
inter-reaction variation in efficiency. Alternatively, an in thermal
standard can be used to adjust the results to account for variation in
amplification efficiency.

[0088] The effect of inter-reaction variability of reaction efficiency
between the reactions used to generate the standard curve and the
reaction used to assay the sample of interest can be eliminated by using
a sample of interest and is amplified with internal standard. The
internal standard is added to reaction in a known copy number and
co-amplified along with the RINF mRNA target. The signal generated from
the known amount of the internal standard provides an indication of the
overall reaction efficiency which can be used to adjust the estimate
difference in copy number to account for the difference in reaction
efficiencies.

[0089] The internal standard is a nucleotide sequence that contains the
same primer binding sites present in the target such that it is amplified
by the same primer pair, but is distinguishable from the target sequence
either by length or, preferably, by the presence of a unique internal
sequence. The internal standard is included in a known copy number
amplifications of the sample of interest and is amplified with
approximately the same efficiency as the target sequence. Any change in
the signal generated by amplification of the internal standard relative
to the signal expected from the standard curve reflects a change in the
overall reaction efficiency and is used to adjust the estimate of the
target sequence copy number correspondingly.

[0090] The fourth aspect of the invention relates to kits comprising
useful components for practicing the present method: A useful kit can
contain oligonucleotide primers and, optionally, probes specific for the
targets regions of the RINF mRNA described. Other optional components of
the kit include, for example, any standard products known by skilled
scientist in the field for the quantitative RT-PCR amplification like,
agents to catalyze the synthesis of primer extension (enzymes, buffer),
the deoxynucleotide triphosphates (dNTP), internal standard nucleic
acids, a reference RNA (positive control), non-amplified control nucleic
acid (negative control), appropriate buffers for PCR or hybridization
reactions, and instructions for carrying out the present method. For
examples, the reference control can be RNA from cell lines such as NB4,
K562, HL60, or MCF7.

[0091] Another kit, or other component in the same kit, can contain SYBR
green, oligonucleotide primers and, optionally, specific for the targets
regions of the RINF. Other components of the kit include, substrate
nucleotide triphosphates, internal standard, agents to catalyze the
synthesis of primer extension, positive control, and negative control.

[0092] The examples of the present invention presented here are provided
only for illustrative purposes and not to limit the scope of the
invention.

Results

[0093] The invention and the results of the experiments leading to the
invention will now be further described, with reference to the following
figures:

[0094] FIG. 1 shows that high RINF mRNA expression is related to bad
prognosis in AML with normal karyotype CN-AML (n=163). In these cohorts,
the cumulative survivals (Kaplan-Meier) are represented according to the
RINF mRNA expression levels detect by affymetrix arrays (HG-U133B or U133
plus 2.0). The patients have been classified in two equivalent groups
(A), according to an expression above (High, n=81) or below (Low, n=81)
the median, or in 3 equivalent groups (B and C), according to a High
(n=55), an Intermediary (n=54), or a Low (n=54) RINF expression level.
Note that because of an odd number of patients (n=163), the patient at
the median has been censored for the 2 groups stratification and were not
exactly equivalents for the 3 groups stratification (n=55 versus 54). P
values (log-rank test) of the comparison of the various groups of
patients are indicated in the panels.

[0095] FIGS. 2 to 5 summarizes the results of the GSE23312 study on AML.
In this study, data for individuals suffering from AML with all
cytogenetic subgroups are available. As shown in FIGS. 2, 3, 4, and 5,
low RINF mRNA expression is related to a better prognosis in all subtypes
of AML, including normal karyotype, as well as with inversion or
translocation (AML-CBF).

[0096] FIGS. 6a and 6b show the prediction of survival outcomes by gene
expression measurement of prognosis factors WT1, MN1, BAALC, ERG, MECOM
(EVI1) in the same cohort of AML patients with normal karyotype CN-AML
(n=163). Gene mRNA expressions were extracted with the specific probesets
WT1 (206067_s_at and/or 206067_s_at), MN1(205330_at), BAALC
(222780_s_at), ERG (241926_s_at) and MECOM (EVI, 226420_at). Cumulative
survivals (Kaplan-Meier) are represented according to mRNA expression
levels detect by affymetrix arrays (HG-U133A, HG-U133B or U133 plus 2.0).
Like for RINF mRNA, the patients have been classified in two equivalent
groups (A), according to an expression above (High, n=81) or below (Low,
n=81) the median, or in 3 equivalent groups (B and C), according to a
High (n=55), an Intermediary (n=54), or a Low (n=54) RINF expression
level. Note that because of an odd number of patients (n=163), the
patient at the median has been censored for the 2 groups stratification
and were not exactly equivalents for the 3 groups stratification (n=55
versus 54). P values (log-rank test) of the comparison of the various
groups of patients are indicated in the panels. Notably, as shown on
FIGS. 2a and 2b, none of these already approved prognosis factors was as
efficient as RINF to predict the survival outcome (as indicated by the P
values) of these AML patients with normal karyotype.

[0097] Moreover, a cohort of ALL patients was tested wether the RINF mRNA
expression could correlate with the risk of relapse. For all the ALL
patients (n=197) of this cohort, RINF expression data were analyzed by
microarray analysis (study GSE13576) as described for the AML cohort
(GSE12417). As shown in FIG. 7, the median RINF mRNA expression was not
equivalent in the different group of patients that relapsed or did not
relapsed. Moreover, as shown in table 1 high RINF mRNA expression is
associated with a higher risk of relapse (11.7% vs. 4.6%), and more
especially of early relapse (9.1% vs. 3%). A high RINF mRNA expression
predicts a three fold increased risk of early relapse compared to low
RINF expression. High and low RINF mRNA expression, respectively,
represents the group of patients with a RINF mRNA expression above or
below the median.

From the above results it is clear that RINF represents a measure
allowing for determining the progression of the disease or a prognosis
for survival of the patient with leukaemia, in particular, acute and
chronic lymphocytic/lymphoblastic or myeloid leukaemia, or the risk of a
relapse or leukaemia in a patient diagnosed and, optionally, treated for
leukaemia. In addition, RINF allows to stratify therapy regimens of
patients afflicted with leukaemia. This is in contrast to other markers
described so far. For example, as shown herein, other markers described
in the art, like MN1 or WT1 do not allow to identify the different groups
of patients afflicted with leukaemia, or at least in a lower extend that
it is possible for RINF and, thus, do not allow to judge on a higher risk
of relapse or on the progression of the disease as well as on the
prognosis for survival and for stratifying the therapy regimens as it is
possible based on RINF. In FIG. 7 RINF mRNA expression (at a diagnosis
time) is higher in the group of acute lymphocytic leukaemia patients
(n=197) that relapse after treatment than in persons showing no relapse.

[0099] Moreover, in chronic myeloid leukaemia based on the data of
GSE4170, RINF expression is increased during the progression of CML from
chronic phase to blast crisis, see FIG. 8. The RINF (CXXC5) gene
expression data were extracted from a gene list (supporting table 4 or
10423Table4.xls) available online at the PNAS website
(http://www.pnas.org/content/103/8/2794/suppl/DC1). CP: chronic phase
(n=42), AP: accelerated phase by blast count criteria (n=9) or by the
occurrence of additional clonal cytogenetic changes (n=8), BC: blast
crisis (n=28). Thus, RINF expression may be used as marker for
identifying progression steps in chronic myeloid but also lymphocytic
leukaemia.

[0100] Quantifying the relative mRNA level of RINF in neoplastic cells
from patients with a leukaemia can help doctors determine whether or not
a tumour is of good or bad prognosis, as well as if a certain treatment
is likely to succeed. In the case of leukaemia, and especially in the
case of AML, these prognostic indicators are important for the physicians
to distinguish the group of patients that would more likely benefit from
a chemotherapy (usually, disease with a favourable prognosis) from
patients that would not and should be considered for stem cell
transplantation (usually, patients with unfavourable prognosis and age
below 60).

[0101] Our results demonstrate that quantification of RINF mRNA expression
could be a significant predictor of patient survival and leukaemia
progression.

[0102] Indeed, by extracting the CXXC5 mRNA expression data from several
independent microarray study available in public databases, and
correlating it with the clinical outcome, we have tested and demonstrated
for the first time, a strong correlation, between a high RINF mRNA
expression and low survival rate for patients suffering from leukaemia,
more precisely AML, ALL, CLL, and CML, more precisely AML with normal
cytogenetics as well as with AML-CBF (translocation t(8;21) or inv(16)).

[0103] At the best of our knowledge, no previous study has examined the
relationship between the RINF mRNA expression and a patient's prognosis
in leukaemia. This inventive idea was tested because we have been the
first to observe that this gene was playing a important role into
differentiation of myeloid cells and was deregulated in cancer cell
types. However, and importantly, these results were not obvious because
many genes, shown to be de-regulated in cancer, have in fact no
predictive value toward a patient survival or response to therapy.
Furthermore, while WO2009/151337 describe that the induction of RINF mRNA
expression (by retinoic acid) correlated with a growth arrest of the
leukemic cell model NB4 in vitro of Acute Promyelocytic Leukemia
(AML-with t(15;17)) (and the clinical remission relapse of the patients).
Here, we surprisingly found the opposite trend in leukemia (AML, ALL,
CLL), since the high RINF expression at diagnosis correlates with a more
agressive phenotype of the leukemic cells (a shorter survival rate of
patients, or a lower probability of response to therapy, or a higher
probability to relapse, or a higher probability to progress into disease
at higher risk).

[0104] Moreover, it was not "obvious" also because we did not find this
trend in an other malignant haemopathies (myeloma) in which High RINF
does not seem to correlate with the survival. This is an important area
of research in cancer because there are little data to help physicians
chose which treatment is best for which patients or to determine when a
change in treatment is warranted.

[0105] The RINF mRNA level can be measured in the biological sample
including blood, liquor, bone marrow, tumour, or tumour biopsy, taken out
at the time of surgery by using the kit and methods according to the
present invention.

[0106] Potentially, this tool could be useful in the clinic for clinicians
to take their decisions about whether a treatment is helping. Our
ultimate goal is to continue effective treatment, but spare patients the
side effects of a treatment that is destined to fail.

[0107] To conclude, the present invention provides a marker with superior
properties over prior art marker allowing to determine the progression of
leukaemia in a patient afflicted therewith or allow to provide a
prognosis for survival of a patient with leukaemia. In addition, the
present invention allows to determine the risk of a relapse of leukaemia
in a patient diagnosed and, optionally, treated for leukaemia based on
RINF expression. Further, the present invention allows to stratify
therapy regimens of individuals afflicted with leukaemia. Moreover, as
demonstrated herein in all four major types of leukaemia, ALL, CLL, AML,
CML, RINF represents a suitable marker, and, strikingly, by comparing
RINF for the first time in the same cohort with other well known markers
in the art, like MN1, WT1, ERG, MECOM or BAALC, RINF has a clearly
superior capability to predict the survival over these other markers.